A healthy intestinal epithelium requires tight coordination of cell proliferation and apoptosis to maintain intact barrier and digestive functions, environmental sampling and immunoregulatory control. Previous work from our laboratory has identified kinase suppressor of Ras (KSR) as an essential mediator of tumor necrosis factor (TNF)-initiated signaling pathways in this tissue. In fact, without KSR kinase activity, intestinal epithelial cells (IECs) exposed to pathological levels of TNF undergo apoptosis. Furthermore, our findings indicate that KSR is required for TNF stimulation of extracellular-regulated-kinases 1 and 2 (ERK1/ERK2), nuclear factor (NF)-kappaB and Akt/protein kinase B. Taken together, these data position KSR as a key regulator of cytokine-mediated cell survival. The goal of this proposal is to test our hypothesis that KSR kinase activity regulates IEC survival during the inflammatory response through activation of anti-apoptotic signal transduction pathways. Supporting a role for KSR function in injury and repair in vivo, we have preliminary data that the KSR+/-IL - 10+/- mouse spontaneously develops inflammatory bowel disease (IBD) with increased apoptosis of colon epithelial cells. Therefore, Aim 1 is designed to determine the mechanisms regulating KSR activation through mutagenesis, tryptic phosphopeptide mapping and in vitro ceramide activation studies. For this Aim, we have developed a novel KSR -/- mouse colon epithelial cell line which will greatly facilitate structure-function analyses. The focus of Aim 2 is to identify substrates and downstream targets of KSR in IECs through in vitro kinase assays, mutational analysis, co-precipitation assays and screening of Cdna expression libraries. In Aim 3 we will study the biological role of KSR in intestinal epithelial injury in vivo using animal models of TNF-induced enteropathy and inflammatory bowel disease (IBD). Because TNF has been implicated in the pathogenesis of a number of gastrointestinal diseases including IBD, necrotizing enterocolitis, celiac disease and non-steroidal anti-inflammatory drug enteropathy, these studies have implications for a number of intestinal conditions resulting from altered programs of cellular proliferation, differentiation and apoptosis. Further, they will determine if KSR is a potential therapeutic target for gastrointestinal inflammation.